PROJECT SUMMARY Obesity and insulin resistance are factors associated with increased risk for and worse outcomes from endometrial cancer (EC). African American (AA) women suffer a higher mortality from EC than Caucasian (CAU) women, and this may be in part due to greater rates of both obesity and diabetes among AA versus CAU patients. Given that the gut microbiome differs by race and has been implicated in the underlying biology of both obesity and cancer, bacteria in the gut and uterus may be logical contributing causes for the racial and obesity disparities seen for EC. A significant gap in our knowledge is that the microbiota of the malignant uterus has not been previously characterized. We hypothesize that the EC microbiota exists, differs by obesity and race status and contributes to the pathogenesis of EC. In our LKB1fl/flp53fl/fl mouse model of endometrioid EC, diet-induced obesity promoted tumor progression, resulting in a doubling of tumor size. Metabolomic profiling revealed significant differences between the ECs in obese versus lean mice, including enhanced energy metabolism and increased gut- microbiome associated metabolites in obese mice. The diabetes drug metformin had heightened efficacy in obese mice, via reversal of the detrimental effects that obesity had on both the gut microbiome and upregulation of energy metabolism. Our pilot study foundational for this grant proposal assessed the microbiota in 21 early stage endometrioid ECs of women and found that bacteria exist in the uterus of the post- menopausal woman. EC microbiota diversity was greater in the tumors of AA versus CAU women, and tumor microbiota profiles were distinct between ECs of obese and lean women. A phase 0 clinical trial of metformin in obese EC patients showed that response to metformin (defined as a decrease in tumor cell proliferation) aligned with greater impact on gut microbiome-associated metabolites. Thus, our preliminary work in both mice and women support a potential critical link between obesity, race, the microbiome and EC. Thus, using our clinically relevant genetically engineered mouse model of obese EC, we will determine in Specific Aim 1 if the microbiota profiles differ between the normal and malignant endometrium in obese and lean post-menopausal, ovariectomized mice. In parallel, in Specific Aim 2 we will expand on our pilot study and assess if the microbiota differ between ECs of non-obese and obese post-menopausal AA and CAU women (N=160 total, 40 per group). Metabolomic profiling will be performed on the ECs from mice and women so as to elicit corresponding function related to obesity- and race-driven differences in the uterine microbiota. Overlay in the techniques of microbiota and metabolomic profiling as well as cross-species comparisons will facilitate discovery of the mechanisms of differing bacterial presence that occur with obesity and AA race in EC. This will potentially lead to microbiota-directed therapies and risk-reducing strategies that will improve outcomes for AA EC patients, which is a public health goal that aligns with the mission of the NIH/NCI.